Recombinant proteins are becoming an important class of therapeutic drugs (1, 2). Many recombinant proteins such as growth hormones and humanized monoclonal antibodies are already in clinical uses (3). One of the limitations for the production of therapeutic proteins in biotech industry is the low yield of the recombinant proteins in cell culture systems. Various approaches have been taken to improve the expression and production of recombinant proteins from transfected mammalian cells, such as the selection of mutants (4), the use of virus-transfected cells (5), or the improvement of the culture medium (6). However, these conventional methods suffer from various shortcomings. For example, the use of mutants means that only certain mutants meeting the expression requirement may be used. This limits the range of proteins that may be expressed. The choice of virus-transfected cells and culture medium are also trial-and-error processes that require laborious experimentations to optimize the conditions. Moreover, they don't always solve problems caused due to structural features of the desired protein.
High quantities of recombinant proteins ranging from hundreds of milligrams to grams must be produced in order to carry out preclinical evaluations and clinical trials (7-9). Unfortunately, potential therapeutic proteins with poor expression face an obstacle to make it through clinical trials to final approval by the FDA. Protein therapeutics developed from recombinant hormones, growth factors and cytokines express at relatively low levels, not only increasing the manufacturing cost but also delaying further product evaluation. Some successful protein therapeutics are recombinant fusion proteins consisting of cytokines or growth factors fused with the Fc portion of IgG1 or immunotoxin and are expressed as single polypeptides with dual biological activities (10,11). These therapeutic fusion proteins, including Enbrel® (TNF-R/Fc-IgG1), Ontak® (IL-2/diphtheria toxin), Orencia® (CTLA-4/Fc-IgG1) and Amevive® (LFA-3/Fc-IgG1) (12), may experience poor expression as the fusion partners interfere with each other for optimal translation, especially in mammalian cells. Since mammalian cells are the preferred choice for producing some therapeutic proteins, as posttranslational modifications in these cells may be associated with reduced immunogenicity compared to other systems (9), a simple strategy that enhances the expression of therapeutic fusion proteins in mammalian cells would be desirable.
Typically, the problem of low expression is improved by incorporating carbohydrate-binding module (CBM) and maltose-binding protein (MBP) as fusion partners to the target protein (13,14). However, these fusion partners are generally removed during or after purification by introducing peptide linkers with cleavage sites for endopeptidases such as thrombin and factor Xa (14). Conceivably, this approach is not feasible for large-scale production of target proteins because it requires numerous steps of column purification and enzymatic processing, limiting the production capacity and possibly causing non-specific cleavage.
The selection of a peptide linker with the ability to maintain domain function of the fusion protein is becoming important (15-18). Recently, the inventors designed a helical linker with 50 amino acids using an EAAAK (SEQ ID NO: 3) helix-forming motif based on a previous study (16), and inserted the linker between granulocyte colony stimulating factor (G-CSF) and Tf moieties, leading to increased biological activity (19). Most recently, the inventors found that the insertion of the same helical linker in Tf-fusion proteins resulted in a high-level expression in HEK293 cells as compared to the same fusion proteins without the helical linker. Here the inventors report the helical linker-dependent increase of expression in two Tf-based fusion proteins, G-CSF and human growth hormone (hGH), and provide evidence of a high-level of expression for both proteins regardless the level of original expression without the linker. Conceivably, this approach can be introduced and applied to other fusion proteins with limited to no expression, greatly improving the production yield for downstream applications.
The above-mentioned and other features of this invention and the manner of obtaining and using them will become more apparent, and will be best understood, by reference to the following description, taken in conjunction with the accompanying drawings. The drawings depict only typical embodiments of the invention and do not therefore limit its scope.